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Homeodomain-interacting protein kinase-2 (HIPK2) can either promote or inhibit transcription depending on cellular context. In this study, we show that a new HIPK2 isoform increases TEAD reporter activity in NSCLC cells. We detected HIPK2 copy number gain in 5/6 (83.3%) NSCLC cell lines. In NSCLC patients with high HIPK2 mRNA expression in the Human Protein Atlas, the five-year survival rate is significantly lower than in patients with low expression (38% vs 47%; p = 0.047). We also found that 70/78 (89.7%) of NSCLC tissues have moderate to strong expression of the N-terminal HIPK2 protein. We detected and cloned a novel HIPK2 isoform 3 and found that its forced overexpression promotes TEAD reporter activity in NSCLC cells. Expressing HIPK2 isoform 3_K228A kinase-dead plasmid failed to increase TEAD reporter activity in NSCLC cells. Next, we showed that two siRNAs targeting HIPK2 decreased HIPK2 isoform 3 and YAP protein levels in NSCLC cells. Degradation of the YAP protein was accelerated after HIPK2 knockdown in NSCLC cells. Inhibition of HIPK2 isoform 3 decreased the mRNA expression of YAP downstream gene CTGF. The specific HIPK2 kinase inhibitor TBID decreased TEAD reporter activity, reduced cancer side populations, and inhibited tumorsphere formation of NSCLC cells. In summary, this study indicates that HIPK2 isoform 3, the main HIPK2 isoform expressed in NSCLC, promotes YAP/TEAD transcriptional activity in NSCLC cells. Our results suggest that HIPK2 isoform 3 may be a potential therapeutic target for NSCLC.

Cyclin-dependent kinase 7 (CDK7) is a protein kinase that plays a major role in transcription initiation. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signalling pathway. Here, we investigated the role of CDK7 on YAP regulation in human malignant pleural mesothelioma (MPM). We found that in microarray samples of human MPM tissue, immunohistochemistry staining showed correlation between the expression level of CDK7 and YAP (n = 70, r = .513). In MPM cells, CDK7 expression level was significantly correlated with GTIIC reporter activity (r = .886, P = .019). Inhibition of CDK7 by siRNA decreased the YAP protein level and the GTIIC reporter activity in the MPM cell lines 211H, H290 and H2052. Degradation of the YAP protein was accelerated after CDK7 knockdown in 211H, H290 and H2052 cells. Inhibition of CDK7 reduced tumour cell migration and invasion, as well as tumorsphere formation ability. Restoration of the CDK7 gene rescued the YAP protein level and GTIIC reporter activity after siRNA knockdown in 211H and H2052 cells. Finally, we performed a co-immunoprecipitation analysis using an anti-YAP antibody and captured the CDK7 protein in 211H cells. Our results suggest that CDK7 inhibition reduces the YAP protein level by promoting its degradation and suppresses the migration and invasion of MPM cells. Cyclin-dependent kinase 7 may be a promising therapeutic target for MPM.

Human non‑small cell lung cancer (NSCLC) is associated with an extremely poor prognosis especially for the 40% of patients who develop brain metastasis, and few treatment strategies exist. Cucurbitacin E (CuE), an oxygenated tetracyclic triterpenoid isolated from plants particularly of the family Cucurbitaceae, has shown anti‑tumorigenic properties in several types of cancer, yet the mechanism remains unclear. Yes‑associated protein (YAP), a main mediator of the Hippo signaling pathway, promotes tumorigenesis, drug resistance and metastasis in human NSCLC. The present study was designed to ascertain whether CuE inhibits YAP and its downstream gene expression in the human NSCLC cell lines H2030‑BrM3 (K‑rasG12C mutation) and PC9‑BrM3 (EGFRΔexon19 mutation), which have high potential for brain metastasis. The efficacy of CuE in suppressing brain metastasis of H2030‑BrM3 cells in a murine model was also investigated. It was found that after CuE treatment in H2030‑BrM3 and PC9‑BrM3 cells, YAP protein expression was decreased, and YAP signaling GTIIC reporter activity and expression of the downstream genes CTGF and CYR61 were significantly (P

: Cullin 4A (Cul4A) is overexpressed in a number of cancers and has been established as an oncogene. This study aimed to elucidate the role of Cul4A in lung cancer invasion and metastasis. We observed that Cul4A was overexpressed in non-small cell lung cancer (NSCLC) tissues and the overexpression of Cul4A was associated with poor prognosis after surgical resection and it also decreased the expression of the tumor suppressor protein annexin A10 (ANXA10). The knockdown of Cul4A was associated with the upregulation of ANXA10, and the forced expression of Cul4A was associated with the downregulation of ANXA10 in lung cancer cells. Further studies showed that the knockdown of Cul4A inhibited the invasion and metastasis of lung cancer cells, which was reversed by the further knockdown of ANXA10. In addition, the knockdown of Cul4A inhibited lung tumor metastasis in mouse tail vein injection xenograft models. Notably, Cul4A regulated the degradation of ANXA10 through its interaction with ANXA10 and ubiquitination in lung cancer cells. Our findings suggest that Cul4A is a prognostic marker in NSCLC patients, and Cul4A plays important roles in lung cancer invasion and metastasis through the regulation of the ANXA10 tumor suppressor.

Although tumour PD-L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD-L1 axis in various cancers, the regulation of PD-L1 (CD274) expression is unclear. Yes-associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down-regulates PD-L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD-L1 protein expression compared to H290, MS-1 and H28 cells. In H2052 and 211H cells, PD-L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD-L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD-L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD-L1 enhancer region encompassing 2 putative YAP-TEAD-binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD-L1 concurrently expressed in immunohistochemistry stain (n = 70, P

Donor organ rejection remains a significant problem. The present study aimed to assess whether transferring a donor's major histocompatibility complex (MHC) genes to the recipient could mitigate rejection in organ transplantation. Seven loci of MHC genes from donor mice were amplified and ligated into vectors; the vectors either contained one K locus, seven loci or were empty (control). The vectors were subsequently injected into the thymus of recipients (in heterotransplants, recipient rats received the vector containing one K locus), following which donor mouse hearts were transplanted. Following the transplantation of allograft and heterograft, electrocardiosignals were viable for a significantly longer duration in recipient mice and rats receiving the donor histocompatibility-2 complex (H-2)d genes compared with those in controls, and in mice that received seven vectors compared with those receiving one vector. Mixed lymphocyte cultures containing cells from these recipients proliferated significantly less compared with mixed lymphocyte cultures containing controls. Also, hearts from H-2d genes-treated recipients demonstrated less lymphocyte infiltration and necrosis compared with the control recipient. The present study concluded that allograft and heterograft rejection may be mitigated by introducing the donor's MHC into the recipient; transferring seven loci has been demonstrated to be more effective than transferring one locus.

Yes-associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030-BrM3(K-rasG12C mutation) and PC9-BrM3 (EGFRΔexon19 mutation) had a significantly decreased p-YAP(S127)/YAP ratio compared to parental H2030 (K-rasG12C mutation) and PC9 (EGFRΔexon19 mutation) cells (P

Programmed death-ligand 1 (PD-L1) is a membrane protein on tumor cells that binds to the PD-1 receptor expressed on immune cells, leading to the immune escape of tumor cells. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signaling pathway, which plays important roles in cancer development. Here we show that YAP regulates PD-L1 expression in human non-small cell lung cancer (NSCLC) cells. First, we investigated YAP and PD-L1 expression at the protein level in 142 NSCLC samples and 15 normal lung samples. In tumor tissue, immunohistochemistry showed positive staining for YAP and PD-L1, which correlated significantly (n = 142, r = 0.514, P < 0.001). Second, in cell lines that express high levels of PD-L1 (H460, SKLU-1, and H1299), the ratio of p-YAP/YAP was lower and GTIIC reporter activity of the Hippo pathway was higher than those in three cell lines expressing low levels of PD-L1 (A549, H2030, and PC9) (P < 0.05). Third, in the same three cell lines, inhibition of YAP by two small interfering RNAs (siRNAs) decreased the mRNA and protein level of PD-L1 (P < 0.05). Fourth, forced overexpression of the YAP gene rescued the PD-L1 mRNA and protein level after siRNA knockdown targeting 3'UTR of the endogenous YAP gene. Finally, chromatin immunoprecipitation (ChIP) assays using a YAP-specific monoclonal antibody resulted in the precipitation of PD-L1 enhancer region encompassing two putative TEAD binding sites. Our results indicate that YAP regulates the transcription of PD-L1 in NSCLC.

A small population of cancer cells called cancer-initiating cells or cancer stem cells (CSCs) are involved in drug resistance, metastasis, and cancer relapse. Finding pathways that regulate CSC is very important for clinical therapy. ATP-binding cassette sub-family G member 2 (ABCG2) plays a role in side population (SP) cell formation and contributes to chemotherapy resistance in common forms of cancer. Yes-associated protein 1 (YAP1) is a major transcriptional effector of the Hippo pathway, which plays important roles in organ size control and tumorigenesis. In this study, we found ABCG2 and YAP1 were both overexpressed in lung cancer SP cells. Disruption of YAP1 expression by siRNA attenuated the expression of ABCG2 transcript and significantly reduced the percentage of SP cells and sphere formation in lung cancer cells. Overexpression of YAP1 in lung cancers led to an increase in ABCG2 expression and increased the percentage of SP cells. However, overexpression of YAP1 in purified non-SP cells did not increase ABCG2 expression and the percentage of SP cells, which may be due to the inhibition of YAP activity through phosphorylation. YAP1 directly transcriptionally regulated ABCG2 by binding to the promoter of ABCG2. Moreover, the YAP1 inhibitor verteporfin and YAP1 siRNA downregulated ABCG2 level through inhibition of YAP1 in lung cancer cells and sensitized them to the chemotherapy drug doxorubicin. Our study adds a new function for YAP1 that may be relevant to drug resistance and cancer therapy through regulation of ABCG2 and side population cell formation in lung cancer.

Yes-associated protein (YAP) is a main mediator of the Hippo pathway, which promotes cancer development. Here we show that YAP promotes resistance to erlotinib in human non-small cell lung cancer (NSCLC) cells. We found that forced YAP overexpression through YAP plasmid transfection promotes erlotinib resistance in HCC827 (exon 19 deletion) cells. In YAP plasmid-transfected HCC827 cells, GTIIC reporter activity and Hippo downstream gene expression of AREG and CTGF increased significantly (P

Cullin 4A (Cul4A) has been observed to be overexpressed in various cancers. In this study, the role of Cul4A in the growth and chemosensitivity in lung cancer cells were studied. We showed that Cul4A is overexpressed in lung cancer cells and tissues. Knockdown of the Cul4A expression by shRNA in lung cancer cells resulted in decreased cellular proliferation and growth in lung cancer cells. Increased sensitivity to gemcitabine, a chemotherapy drug, was also noted in those Cul4A knockdown lung cancer cells. Moreover, increased expression of p21, transforming growth factor (TGF)-β inducible early gene-1 (TIEG1) and TGF beta-induced (TGFBI) was observed in lung cancer cells after Cul4A knockdown, which may be partially related to increased chemosensitivity to gemcitabine. G0/G1 cell cycle arrest was also noted after Cul4A knockdown. Notably, decreased tumour growth and increased chemosensitivity to gemcitabine were also noted after Cul4A knockdown in lung cancer xenograft nude mice models. In summary, our study showed that targeting Cul4A with RNAi or other techniques may provide a possible insight to the development of lung cancer therapy in the future.

BackgroundMaintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. PR-Set7 (also known as Set8) is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20 (H4K20me1) to promote chromosome condensation and prevent DNA damage. Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage. This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage.MethodsWe developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A. We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo. With this model, staining of PR-Set7 in the preneoplastic and tumor lesions in AdenoCre-induced mouse lungs was performed. Meanwhile we identified higher protein level changes of γ-tubulin and pericentrin by IHC.ResultsThe level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A. We also identified higher levels of the proteins pericentrin and γ-tubulin in Cul4A mouse lungs induced by AdenoCre.ConclusionsPR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.

BackgroundThe Hedgehog (Hh) signaling pathway has been implicated in stem cell maintenance and its activation is aberrant in several types of cancer including mesothelioma. Protein kinase CK2 affects several cell signaling pathways through the mechanism of phosphorylation.MethodsProtein and mRNA levels of CK2α and Gli1 were tested by quantitative RT-PCR and immunohistochemistry staining in mesothelioma samples and cell lines. Down-regulated Gli1 expression and transcriptional activity were demonstrated by RT-PCR, Western blot and luciferase reporter assay.ResultsIn this study, we show that CK2α is over-expressed and a positive regulator of Hegdehog/Gli1 signaling in human malignant pleural mesothelioma. First of all, we found that the mRNA levels of CK2α and Gli1 were broadly elevated and correlated (n = 52, r = 0.401, P